View - ResearchGate

26 Charlton and Zachariou
Since the first set of work was published describing the immobilization of
metal ions using a chelating agent covalently attached to a stationary support
to purify proteins (3,4), there have been several modifications and adaptations
of this technique over the years. The fundamental approach remains to use
immobilized metal ions, and, in particular borderline Lewis metal ions such
as Cu 2+ ,Ni 2+ and Zn 2+ , to purify proteins on the basis of their histidine
content (3).
In 1985, there were indications that electrostatic interactions were also
occurring between proteins and immobilized Fe 3+ -iminodiacetic acid (IDA)
stationary phases (5), and in 1996, it was demonstrated that IMAC adsorbents
in general could also be used in pseudo-cation exchange mode, independently
of histidine interaction (6). Yet another mode of interaction involved in the
IMAC of proteins was the mixed mode interactions involving aspartate and/or
glutamate surface residues on proteins along with electrostatic interactions,
again independent of histidine interactions (7). It is the purpose of this work to
describe the methodologies involved in the traditional histidine-based IMAC
interactions, the mixed mode interactions involving aspartate, glutamate and
electrostatic interactions and then the purely electrostatic interactions. The
reader is referred to reviews of IMAC of proteins for a more detailed perspective
(8,9,10,11).
The traditional use of IMAC for proteins has been to select proteins on the
basis of their histidine content. The approach uses a chelating agent immobilized
on a stationary surface to capture a metal ion and form an immobilized metal
chelate complex (IMCC). The chelating agent has usually been the tridentate
IDA, despite a plethora of chelating stationary supports available for such work
(12). Generally, Cu 2+ ,Ni 2+ and Zn 2+ have been used in this mode, but other
metal ions such as Co 2+ ,Cd 2+ ,Fe 2+ and Mn 2+ have also been examined as the
metal ions of choice. Histidine selection by the IMCC exploits the preference
of borderline Lewis metals (see ref. 13 for a review of the concept of hard and
soft acids and bases and their preferred interactions) to accept electrons from
borderline Lewis bases such as histidine. With a pKa of 6, histidine will be able
to donate electrons effectively at pH > 6.5 and thus bind to the IMCC, although
this may vary depending on the microenvironment the histidine finds itself
in. Once the protein has bound, a specific elution can be deployed by using
imidazole, which is the functional moiety of histidine. Alternatively, the pH
may be decreased to